Use of Flagellin in Tumor Immunotherapy

ABSTRACT

The invention provides a fusion protein comprising a flagellin adjuvant and a tumor antigen. Also provided are compositions comprising a flagellin adjuvant and a tumor antigen. The invention further provides pharmaceutical formulations and methods for inducing an immune response against a tumor antigen and methods of treating a tumor in a subject.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/636,635, filed 16 Dec. 2004, and U.S. Provisional ApplicationSer. No. 60/709,609, filed 19 Aug. 2005; the disclosures of which areincorporated herein by reference in their entireties.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under grant numbersR01-A1051319 and T32-AI007401 from the National Institutes of Health.The United States government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns the use of a flagellin adjuvant, tumorantigens and fusion proteins thereof to produce an immune responseagainst tumors (e.g., in prophylactic or therapeutic methods).

BACKGROUND OF THE INVENTION

Currently accepted treatments for breast cancer include surgery,chemotherapy, and radiation therapy. Immunotherapy represents arelatively new approach for which there has been a surge of interest(Ribas et al. (2003) J. Clin. Oncol. 21:2415-2432; Scanlan et al. (2001)Breast Cancer Res. 3:95-98; Jäger et al. (2002) Curr. Opin. Immunol.14:178-182; Ko et al. (2003) Clin. Cancer Res. 9:3222-3234). The majoradvantages of an immunological approach are that it is not invasive andit selectively targets breast cancer cells, leaving normal cellsunharmed. In recent years, investigators have defined a number ofproteins that are mutated or inappropriately expressed by breast cancercells that may serve as useful targets for immunotherapy. For example,phase I/II clinical trials have been initiated using mucin (MUC-1),HER-2/neu, CEA, and hTERT vaccines (see Ko et al. (2003) Clin. CancerRes. 9:3222-3234 for review). Although a number of these studies haverevealed instances of enhanced immune reactivity, for example, T cellproliferation or cytotoxic T cell activity, positive clinical resultshave been limited to a relatively small subset of patients.

The lack of strong clinical responses to tumor vaccines is likely due toa combination of several factors. Since some of the target tumorantigens are over-expressed, but not mutated, it is possible thatself-tolerance precludes a strong response. However, this may not be thecase with mutated forms of these antigens. Alternatively, the protocolsthat have been used to immunize patients may promote the generation oflow, but not high avidity T cells (see Ko et al. (2003) Clin. CancerRes. 9:3222-3234 for discussion); the high avidity cells being mosteffective in tumor cell clearance. Finally, the over-production oftransforming growth factor β (TGF-β) by tumor cells may limit theeffectiveness of a potentially efficacious vaccination strategy.

Work in other systems has established the value of adjuvants in theenhancement of weak immune responses. Thus it is possible that a potentadjuvant may allow the immune system to not only overcome theimmunosuppressive effect of tumor-derived TGF-β, but also promote thegeneration of high avidity anti-tumor T cells, NK cells, or antibodies.It should be noted that a number of vaccine protocols have used avariety of adjuvants ranging from individual cytokines (e.g., IL-2,IL-12, gamma interferon, or GM-CSF) to microbial cell components (e.g.,Detox B or BCG). Although individual cytokines possess some adjuvantactivity, their effectiveness may be limited by the scope of theirindividual biologic activities. There is clearly a need to develop andtest new adjuvants that promote more effective anti-tumor responses.

It would be desirable to provide improved reagents, pharmaceuticalformulations and methods for producing immune responses against cancer.

SUMMARY OF THE INVENTION

A first aspect of the invention is a fusion protein comprising,consisting of, or consisting essentially of: (a) a flagellin adjuvantcomprising, consisting of, or consisting essentially of (i) a flagellinN-terminal constant region; (ii) a flagellin C-terminal constant region;and (b) a tumor antigen between the N-terminal constant region and theC-terminal constant region (e.g., inserted into or in place of part orall of the flagellin hypervariable region, which is optionally partiallyor entirely deleted).

A further aspect of the invention is a nucleic acid encoding a fusionprotein as described above. In some embodiments, the nucleic acid isoperably associated with a promoter.

A further aspect of the invention is a vector comprising a nucleic acidas described above.

A further aspect of the invention is a host cell comprising a nucleicacid or vector as described above. In some embodiments, the host cellexpresses the encoded fusion protein.

A further aspect of the invention is a method of making a fusion proteinas described above, the method comprising culturing a host cellcomprising a nucleic acid encoding the fusion protein described above ina suitable culture medium under conditions sufficient for the fusionprotein to be production. Optionally, the fusion protein is collectedfrom the host cell or from the culture medium.

A further aspect of the invention is a composition comprising,consisting of, or consisting essentially of (a) a flagellin adjuvant,and (b) a tumor antigen (with the tumor antigen and the flagellinadjuvant being separate or coupled to one another, i.e., in the form ofa fusion protein, for example, a fusion protein as described herein).

A further aspect of the present invention is a pharmaceuticalformulation comprising a fusion protein or a composition as describedabove in a pharmaceutically acceptable carrier.

A further aspect of the invention is a method of inducing an immuneresponse (e.g., producing antibodies and/or inducing a cell-mediatedimmune response) to a tumor antigen in a subject, comprisingadministering a fusion protein, a composition, or a pharmaceuticalformulation as described above to the subject in an amount effective toinduce an immune response to the tumor antigen in the subject.

A still further aspect of the invention is a method of treating a tumorin a subject, the method comprising administering a fusion protein, acomposition, or a pharmaceutical formulation as described above to thesubject in a treatment effective amount. The invention can be practicedin therapeutic and prophylactic methods.

In particular embodiments of the methods of the invention, the subjectis a mammalian subject, a primate subject, or a human subject.

A further aspect of the invention is the use of a fusion protein orcomposition as described herein for the preparation of a medicament forcarrying out a method of treatment as described herein.

These and other aspects of the invention are set forth in thedescription of the invention that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts TNFα expression in the lungs of BALB/c mice givenflagellin or mutant flagellin 229 by i.t. instillation.

FIG. 2 depicts the effect of flagellin on tumor growth. The open circlesrepresent the mice given the Fra-1 antigen and the inactive form offlagellin. The closed circles represent the mice given Fra-1 antigen andthe active form of flagellin.

FIG. 3 shows that immunization with flagellin and the F1 antigen ofYersinia pestis results in substantial anti-F1 antibody production.(Panel a) Female BALB/c mice were immunized intratracheally (i.t.) orintranasally (i.n.) with 10 μg F1+1 μg flagellin (FliC). Control animalswere immunized i.t. with 10 μg F1 alone or with 1 μg of the 229 mutantflagellin. Mice were boosted in an identical manner at 4 weeks andplasma was collected 2 weeks later for analysis by ELISA. Numbers withinthe bars indicate ratio of IgG1/IgG2a isotypes. * indicates statisticalsignificance over controls and ** indicates i.n. titers arestatistically greater than i.t. (p<0.007). (Panel b) Anti-F1 antibodytiters from mice immunized i.n. with 10 μg F1+1 μg flagellin. Each linerepresents one mouse and arrows indicate booster immunizations. (Panelc) Female BALB/c mice were immunized i.t. with 10 μg F1 and increasingamounts of FliC or 5 μg of 229 and boosted at 4 weeks. Plasma anti-F1IgG titers were determined 2 weeks post-boost. (Panel d) A group offemale BALB/c mice was immunized i.n. with 5 μg of flagellin alone andboosted in an identical manner at 4 weeks. Anti-FliC antibody titerswere determined 2 weeks later (mean anti-FliC titer=8.5×10⁵) andflagellin-immune mice were then immunized and boosted with 10 μg F1+1 μgFliC i.n. Two weeks post-boost, anti-F1 titers were determined andcompared to titers of flagellin-naive animals immunized with 10 μg F1+1μg FliC or 229. Bars represent mean antibody titers ±s.e.m. Seven femaleBALB/c mice were used per immunization group.

FIG. 4 shows that flagellin stimulates antigen-specific responses andrequires T cells. (Panel a) Groups of 7 female BALB/c mice wereimmunized i.n. with 10 μg F1 antigen+1 μg flagellin (FliC) and boostedat 4 weeks with PBS, 1 μg FliC alone, 10 μg F1 alone or 10 μg F1+1 μgFliC. Plasma was collected 3 weeks after boosting for analysis byELISA. * indicates statistical significance over animals boosted withPBS or FliC alone and ** indicates that boosting with F1+FliC results inantibody titers statistically greater than F1 antigen alone (p<0.01).Bars represent mean antibody titers ±s.e.m. (Panel b) A group of 7athymic nude mice (BALB/cAnNCr-nu/nu) was immunized and boosted i.n.with 10 μg F1+1 μg FliC. Plasma was collected 2 weeks post-boost foranalysis of anti-F1 IgG titers by ELISA. * indicates statisticalsignificance compared to normal BALB/c mice immunized in an identicalmanner (p<0.001).

FIG. 5 depicts the requirements for the adjuvant effects of flagellin.TNFR^(−/−) (panel a) or IL6^(−/−) (panel b) and wild-type B6; 129control mice were immunized i.t. with 10 μg F1 antigen+1 μg flagellin(FliC) or mutant flagellin (229). * indicates TNFR^(−/−) titers arestatistically less than B6; 129 control (p<0.001). C3H/HeJ (Tlr4 P712Hmutant) and wild-type C3H/HeN mice were immunized with 10 μg F1+1 μgFliC or 229 (panel c). IFNα/βR^(−/−) (panel d) and IFNγ^(−/−) (panel e)mice and corresponding wild-type controls were immunized i.n. with 10 μgF1+1 μg FliC or 229. Seven female mice were used in each immunizationgroup. Mice were boosted in the same manner at 4 weeks and plasma wascollected 2 weeks later for analysis of anti-F1 IgG titers by ELISA.Bars represent mean antibody titers ±s.e.m.

FIG. 6 shows that flagellin promotes a protective response forintranasal infection with Yersinia pestis CO92. Groups of 15 femaleBALB/c mice (panel a) were immunized i.n. with 10 μg F1 antigen+1 μgflagellin (FliC) or PBS alone and boosted in an identical manner at 4weeks. Plasma was collected 2 weeks post-boost for analysis of antibodytiters by ELISA (mean anti-F1 titer=9.4×10⁵). One week later mice werechallenged i.n. with a dose of Y. pestis CO92 equivalent to 100×LD₅₀.Mice were monitored for 30d post-challenge. Groups of 10antibody-deficient IgH^(−/−) mice (panel b) were immunized i.n. with 10g F1+1 μg FliC or PBS alone and boosted in an identical manner at 4weeks. Mice were challenged two weeks later i.n. with a dose of Y.pestis equivalent to 155×LD₅₀. Groups of 10 wild-type C57BL/6 mice(panel c) and female IFNγ^(−/−) mice (panel d) were immunized andboosted i.n. with 10 μg F1+1 μg FliC. Plasma was collected 2 weekspost-boost for analysis of antibody titers by ELISA (anti-F1titer≧1×10⁶). One week later mice were challenged i.n. with a dose of Y.pestis equivalent to 150×LD₅₀ and monitored for 16d post-challenge

FIG. 7 shows that flagellin is an effective adjuvant in nonhumanprimates. Female cynomolgus monkeys (Macaca fascicularis) were immunizedintranasally (n=6) or intramuscularly (n=6) with 150 μg F1/V fusionprotein+50 μg flagellin. Control animals (n=3) were immunized i.n. andi.m. with PBS alone. No significant change in body temperature occurredover 12 h and TNF-α was not detected in plasma collected at 4 h, 12 hand 24 h post-immunization. Animals were boosted in an identical mannerat 4 weeks and plasma was collected 2 weeks later for analysis by ELISA.Bars indicate mean anti-F1/V antibody titers ±s.e.m. and * indicatesstatistical significance over intranasal immunization (p<0.006).

FIG. 8 shows that a fusion protein containing flagellin and the Yersiniapestis F1 and V proteins retains flagellin biological activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based, in part, on the discovery thatflagellins, and fragments thereof, can function as adjuvants to enhancethe anti-tumor immune response mounted in a subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

By “consisting essentially of” as used herein, it is meant that theindicated peptide, protein, fusion protein, nucleic, compound,composition, and the like does not include any other material elements(i.e., elements that materially impact the structure and/or function ofthe peptide, protein, fusion protein, nucleic acid, compound orcomposition).

In representative embodiments of the invention, the peptides, proteins,fusion proteins, nucleic acids and/or cells of the invention are“isolated.” By “isolated” it is meant that the peptide, protein, fusionprotein, nucleic acid and/or cell is at least partially purified awayfrom other components.

1. Tumor Antigens.

The term “tumor antigen” as used herein indicates a molecule (e.g., aprotein or peptide) that is expressed by a tumor cell and either (a)differs qualitatively from its counterpart expressed in normal cells, or(b) is expressed at a higher level in tumor cells than in normal cells.Thus, a tumor antigen can differ from (e.g., by one or more amino acidresidues where the molecule is a protein), or it can be identical to,its counterpart expressed in normal cells. Some tumor antigens are notexpressed by normal cells, or are expressed at a level at least abouttwo-fold higher (e.g., about two-fold, three-fold, five-fold, ten-fold,20-fold, 40-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or15,000-fold higher) in a tumor cell than in the tumor cell's normalcounterpart.

Any suitable tumor antigen can be used in the practice of the presentinvention. Tumor antigens include without limitation naturally occurringtumor antigens and modified forms thereof that induce an immune responsein a subject, and further include antigens associated with tumor cellsand antigens that are specific to tumor cells and modified forms of theforegoing that induce an immune response in a subject. The term tumorantigen further encompasses antigens that correspond to proteins thatare correlated with the induction of tumors such as oncogenic virusantigens (e.g., human papilloma virus antigens). Exemplary tumorantigens include, without limitation, HER2/neu and BRCA1 antigens forbreast cancer, MART-1/MelanA (melanoma antigen), Fra-1 (breast cancer),NY-BR62, NY-BR85, hTERT, gp100, tyrosinase, TRP-1, TRP-2, NY-ESO-1,CDK-4, β-catenin, MUM-1, Caspase-8, KIAA0205, SART-1, PRAME, and p15antigens, members of the MAGE family (melanoma antigens), the BAGEfamily (melanoma antigens), the DAGE/PRAME family (such as DAGE-1), theGAGE family (melanoma antigens), the RAGE family (such as RAGE-1), theSMAGE family, NAG, TAG-72, CA125, mutated proto-oncogenes such asp21ras, mutated tumor suppressor genes such as p53, tumor associatedviral antigens (e.g., HPV E6 and E7), the SSX family, HOM-MEL-55,NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11,HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-1, and the SCP family.Members of the MAGE family include, but are not limited to, MAGE-1,MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-11 and MAGE-12. Members of the GAGEfamily include, but are not limited to, GAGE-1, GAGE-6. See, e.g., thereview by Van den Eynde and van der Bruggen, (1997) Curr. Opin. Immunol.9: 684-693; and Sahin et al., (1997) Curr. Opin. Immunol. 9: 709-716.

The tumor antigen can also be, but is not limited to human epithelialcell mucin (Muc-1; a 20 amino acid core repeat for the Muc-1glycoprotein, present on breast cancer cells and pancreatic cancercells), MUC-2, MUC-3, MUC-18, carcino-embryonic antigen (CEA), the rafoncogene product, CA-125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2,prostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), GnT-V intron V sequence (N-acetylglucosaminyltransferase Vintron V sequence), Prostate Ca psm, MUM-1-B (melanoma ubiquitiousmutated gene product), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72,β-HCG, gp43, HSP-70, p17 mel, HSP-70, gp43, HMW, HOJ-1, melanomagangliosides, TAG-72, mutated proto-oncogenes such as p21ras, mutatedtumor suppressor genes such as p53, estrogen receptor, milk fatglobulin, telomerases, nuclear matrix proteins, prostatic acidphosphatase, protein MZ2-E, polymorphic epithelial mucin (PEM),folate-binding-protein LK26, truncated epidermal growth factor receptor(EGFR), Thomsen-Friedenreich (T) antigen, GM-2 and GD-2 gangliosides,polymorphic epithelial mucin, folate-binding protein LK26, humanchorionic gonadotropin (HCG), pancreatic oncofetal antigen, cancerantigens 15-3, 19-9, 549, 195, squamous cell carcinoma antigen (SCCA),ovarian cancer antigen (OCA), pancreas cancer associated antigen (PaA),EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, chimeric proteinp210_(BCR-ABL), lung resistance protein (LRP) Bcl-2, and Ki-67. See,e.g., U.S. Pat. No. 6,537,552; see also U.S. Pat. Nos. 6,815,531;6,773,707; 6,682,928; and 6,623,739.

The tumor antigen can also be an antibody produced by a B cell tumor(e.g., B cell lymphoma; B cell leukemia; myeloma; hairy cell leukemia),a fragment of such an antibody, which contains an epitope of theidiotype of the antibody, a malignant B cell antigen receptor, amalignant B cell immunoglobulin idiotype, a variable region of animmunoglobulin, a hypervariable region or complementarity determiningregion (CDR) of a variable region of an immunoglobulin, a malignant Tcell receptor (TCR), a variable region of a TCR and/or a hypervariableregion of a TCR. In one embodiment, the tumor antigen of this inventioncan be a single chain antibody (scFv), comprising linked V_(H), andV_(L) domains, which retains the conformation and specific bindingactivity of the native idiotype of the antibody.

Certain tumor antigens referred to as “cancer/testis” antigens arerelatively prevalent on a wide array of different tumors. Thus, use ofthe present invention to administer one or more cancer/testis antigenscan elicit a response against an array of tumors. For example, acocktail comprising a flagellin adjuvant and a series of (e.g., two ormore, for example 6-12) cancer/testis antigens (e.g., in the form of afusion protein of the invention) can be used to treat a wide array oftumors, possibly avoiding the need to identify particular tumor antigensexpressed by a subject's tumor on an individualized basis. Non-limitingexamples of cancer/testis antigens include but are not limited toMAGE-A, MAGE-B, BAGE, GAGE-A, SSX-2, NY-ESO-1, SCP-1, CT7/MAGE-C1HOM-TES-85, CT/BRDT, CT10, CTp11/SPAN-X-C1, SAGE, OY-TES-1, cTAGE-I,CT15/Fertilin beta, CT16, CT17, MMA-I and CAGE, which can be usedindividually or in any combination with each other and/or with othertumor antigens.

Also encompassed by the present invention are modified forms of thetumor antigens described above which induce an immune response in asubject. Modified forms of naturally occurring tumor antigens canadvantageously have reduced pathogenicity and/or enhanced immunogenicityas compared with the naturally occurring antigen.

Fragments of naturally-occurring tumor antigens that induce an immuneresponse in the subject can be used according to the present invention.The fragment can comprise one or multiple epitopes, and can furthercomprise 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or moreconsecutive amino acids of the full-length protein. In embodiments ofthe invention, the fragment comprises all or substantially all (e.g.,all but about 1, 2, 3, 5, 10, 15, 20, 25 or 50 amino acids) of theextracellular domain of the protein, optionally without thetransmembrane and/or cytoplasmic portions. In other embodiments, thefragment comprises at least about 6, 10, 15, 20, 30, 40, 50, 75, 100,250, 500 or more consecutive amino acids of the extracellular portion ofthe protein, optionally without the transmembrane or cytoplasmicportions.

Further, the recitation of a “NY-ESO-1 antigen,” or any other specifiedtumor antigen, includes without limitation any naturally occurringHer2/neu antigen (or other specified tumor antigen), and modified formsthereof that induce an immune response in a subject.

For example, any suitable NY-ESO-1 antigen can be used with the presentinvention, including the full-length protein and fragments thereof. Forexample, the antigen can comprise one or multiple epitopes, and canfurther comprise 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or moreconsecutive amino acids of the NY-ESO-1 protein. In particularembodiments, the NY-ESO-1 antigen is as described in U.S. PatentPublication No. 2004/0203051 A1. In further embodiments, the NY-ESO-1antigen comprises all or substantially all (e.g., all but about 1, 2, 3,5, 10, 15, 20, 25 or 50 amino acids) of the extracellular domain of theprotein, optionally without the transmembrane and/or cytoplasmicportions. In other embodiments, the NY-ESO-1 antigen comprises at leastabout 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or more consecutiveamino acids of the extracellular portion of the NY-ESO-1 protein,optionally without the transmembrane or cytoplasmic portions. As will beappreciated by those skilled in the art, the NY-ESO-1 antigen can bepresented in the form of a fusion peptide (alone or as part of a fusionprotein of the invention). For example, a fusion peptide comprising theNY-ESO-1 antigen and a cytokine (e.g., interferon-γ, IL-2 [includingIL-2 immunotoxins, such as IL-2-diphtheria toxin], IL-12, or GM-CSF) canbe delivered. Also encompassed by the present invention are modifiedforms of any of the foregoing that induce an immune response in asubject.

Any suitable Her2/neu antigen can be used with the present invention,including the full-length protein and fragments thereof. For example,the antigen can comprise one or multiple epitopes, and can furthercomprise 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or moreconsecutive amino acids of the Her2/neu protein. In particularembodiments, the Her2/neu antigen is as described in U.S. PatentPublication No. 20040241686. In other embodiments, the Her2/neu antigencomprises an epitope as described in U.S. Patent Publication No.20040121946. In further embodiments, the Her2/neu antigen comprises allor substantially all (e.g., all but about 1, 2, 3, 5, 10, 15, 20, 25 or50 amino acids) of the extracellular domain of the protein, optionallywithout the transmembrane and/or cytoplasmic portions, see, e.g., U.S.Pat. No. 6,333,169. In other embodiments, the Her2/neu antigen comprisesat least about 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or moreconsecutive amino acids of the extracellular portion of the Her2/neuprotein, optionally without the transmembrane or cytoplasmic portions.As will be appreciated by those skilled in the art, the Her2/neu antigencan be presented in the form of a fusion peptide (alone or as part of afusion protein of the invention). For example, a fusion peptidecomprising the Her2/neu antigen and a cytokine (e.g., IL-12, IL-2[including IL-2 immunotoxins, such as IL-2-diphtheria toxin] or GM-CSF)can be delivered (see, e.g., Dela Cruz et al., (2003) Vaccine 21: 1317).Also encompassed by the present invention are modified forms of any ofthe foregoing that induce an immune response in a subject.

The tumor antigens that can be used in accordance with the presentinvention are in no way limited to the tumor antigens listed herein.Other tumor antigens can be identified, isolated and cloned by methodsknown in the art such as those disclosed in U.S. Pat. No. 4,514,506.

It has recently been shown that regulatory T cells play a major role inlimiting the effectiveness of anti-tumor vaccines. Strategies that block(generally temporarily) regulatory T cell activity (e.g., via an IL-2immunotoxin, such as an IL-2-diphtheria toxin, or γ-interferon) can beused to enhance the effectiveness of the fusion proteins, compositionsand methods of the invention. The IL-2 immunotoxin or γ-interferon canbe can be formulated and administered with the fusion proteins andimmunogenic compositions of the invention. Alternatively, they can beformulated separately and are optionally administered concurrently withthe fusion proteins and immunogenic compositions of the invention. Asused herein, the term “concurrently” means sufficiently close in time toproduce a combined effect (that is, concurrently can be simultaneously,or it can be two or more events occurring within a short time period[e.g., minutes or hours] before or after each other). In particularembodiments, a flagellin/tumor antigen fusion protein comprises the IL-2immunotoxin or γ-interferon. In other embodiments wherein the flagellinadjuvant and the tumor antigen are not provided as a fusion protein, theIL-2 immunotoxin or γ-interferon is fused to the flagellin or the tumorantigen.

The term “tumor” is understood in the art, for example, as an abnormalmass of undifferentiated cells within a multicellular organism. Tumorscan be malignant or benign. Generally, the inventive methods disclosedherein are used to treat malignant tumors. The tumor to be treated orimmunized against (i.e., prophylactic treatment) can be, but is notlimited to, tumors present in myeloma, hematological cancers such asleukemias and lymphomas (such as B cell lymphoma, T cell lymphoma,Hodgkin's lymphoma, non-Hodgkins lymphoma), hematopoietic neoplasmas,thymoma, head and neck cancer, sarcoma, lung cancer, liver cancer,genitourinary cancers (such as ovarian cancer, vaginal cancer, cervicalcancer, uterine cancer, bladder cancer, testicular cancer, prostatecancer or penile cancer), adenocarcinoma, breast cancer, pancreaticcancer, lung cancer, renal cancer, liver cancer, primary or metastaticmelanoma, squamous cell carcinoma, basal cell carcinoma, neurologicaltumors including brain tumors such as astrocystomas and glioblastomas,angiosarcoma, hemangiosarcoma, head and neck cancer, thyroid carcinoma,soft tissue sarcoma, bone cancer such as bone sarcoma, vascular cancer,gastrointestinal cancer (such as gastric, stomach or colon cancer), andany other tumor now known or later identified (see, e.g., Rosenberg(1996) Ann. Rev. Med. 47:481-491.

2. Flagellins.

The inventor has determined that flagellin can function as an adjuvantto enhance the active immune response mounted by a host to a tumorantigen. As used herein, the term “adjuvant” has its ordinary meaning asunderstood by those skilled in the art. For example, an adjuvant can bedefined as a substance that increases the ability of an antigen tostimulate an immune response against the antigen in the subject. Inparticular embodiments, the adjuvant increases the immune responseagainst the antigen by at least about 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60, 75, 100, 150, 500, 1000-fold or more. In other embodiments, theadjuvant reduces the amount of antigen required to achieve a particularlevel of immune response (cellular and/or humoral and/or mucosal), e.g.,a reduction of at least about 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%,90%, 95%, 98% or more. An adjuvant can further be a substance thatprolongs the time over which an immune response, optionally protectiveimmune response, is sustained (e.g., by at least about a 2-fold, 3-fold,5-fold, 10-fold, 20-fold longer time period or more). In some instancesthere may be no significant immune response elicited in the host in theabsence of an adjuvant.

Flagellin proteins are known and described, for example, in U.S. Pat.Nos. 6,585,980, 6,130,082; 5,888,810; 5,618,533; 4,886,748 and U.S.Patent Publication No. US 2003/0044429 A1; and Donnelly et al., (2002)J. Biol. Chem. 43: 40456. Most gram-negative bacteria express flagella,which are surface structures that provide motility. The flagella areformed from a basal body, a filament, and a hook that connects the two.The filament is formed of a long polymer of a single protein, flagellin,with a small cap protein at the end. Polymerization of flagellin ismediated by conserved regions at the N- and C-termini, whereas theintervening regions of the flagellin protein are very diverse amongspecies.

In illustrative embodiments of the invention, a fusion protein isprovided comprising a flagellin adjuvant and one or more tumor antigens.In general, fusion proteins of the invention comprise, consistessentially of, or consist of: (a) a flagellin adjuvant comprising (i) aflagellin N-terminal constant region; and (ii) a flagellin C-terminalconstant region; and (b) a tumor antigen, wherein the tumor antigen isbetween the N-terminal constant region and the C-terminal constantregion. In some embodiments, the flagellin hypervariable region betweenthe constant regions is deleted (in whole or in part); in otherembodiments the hypervariable region is present. When the hypervariableregion is present (in whole or in part) the antigen can be inserted (i)within the hypervariable region, (ii) between the flagellin N-terminalconstant region and the hypervariable region, or (iii) between theflagellin C-terminal constant region and the hypervariable region.

Further, the N-terminal constant and C-terminal constant regions can belinked by a hinge region. The hypervariable region or a tumor antigencan function as a hinge region. Additionally, or alternatively, asegment of about 2, 3, 4, 6, 8, 10, 15, 20, 30, 50 or more amino acidscan function as a hinge region.

Conserved regions of flagellin are well known in the art and have beendescribed, for example, in Mimori-Kiyosue et al., (1997) J. Mol. Virol.270:222-237; lino et al., (1977) Ann. Rev. Genet. 11:161-182; andSchoenhals et al, (1993) J. Bacteriol. 175:5395-5402. As is understoodby those skilled in the art, the size of the constant region will varysomewhat depending on the source of the flagellin protein. In general,the N-terminal constant domain includes the approximately 170 or 180N-terminal amino acids of the protein, whereas the C-terminal constantdomain typically spans the approximately 85 to 100 C-terminal aminoacids. The central hypervariable region varies considerably by size andsequence among bacteria, and accounts for most of the difference inmolecular mass. The N- and C-terminal constant regions of flagellinproteins from a variety of bacteria are known, and others can be readilyidentified by those skilled in the art using known alignment techniques,which are facilitated by the elucidation of the crystal structure of theflagellin monomer (Samatey et al., (2001) Nature 41:331).

The terms “flagellin N-terminal constant region” and “flagellinC-terminal constant region” as used herein includes active fragments(e.g., fragments of at least about 50, 100 or 120 amino acids in length)and modifications of any of the foregoing that enhance the immuneresponse to the tumor antigen (e.g., by activating the TLR5 pathway).For example, the native flagellin regions can be modified to increasesafety and/or immune response. In some embodiments, the flagellinN-terminal and/or C-terminal constant region comprises the full-lengthregion or, alternatively, can comprise only a fragment of one or bothregions.

In particular embodiments, the N-terminal and/or C-terminal constantregion comprises a TLR5 recognition site(s) and is able to activate theTLR5 pathway.

In representative embodiments, the N-terminal constant region comprisesthe N-terminal RINSA domain (amino acids 31-52 of the S. dublinflagellin) as described by Eaves-Pyles et al. (2001) J. Immunology 167:7009-7016, or a homolog or modified form thereof that enhances theimmunogenicity of the tumor antigen. In other embodiments, theN-terminal constant region comprises the D1 and D2 domains, and theC-terminal constant region comprises the D1 and D2 domains (Eaves-Pyleset al. (2001) J. Immunology 167: 7009-7016) or a modified form thereofthat enhances the immunogenicity of the tumor antigen.

In other embodiments, the flagellin N-terminal and/or C-terminalconstant region comprises, consists of, or consists essentially of thepeptide GAVQNRFNSAIT (SEQ ID NO:4) as described by U.S. PatentPublication No. US 2003/0044429 A1 to Alderem et al., or a homolog ormodification thereof that enhances the immunogenicity of the tumorantigen.

In still other embodiments, the N-terminal constant domain comprises the“motif N” (e.g., amino acids 98-108 of the S. muenchen flagellin) and/orthe C-terminal constant domain comprises the “motif C” (e.g., aminoacids 441-449 of the S. muenchen flagellin) identified by Kanneganti etal., (2004) J. Biol. Chem. 279:5667-5676, or a homolog or modified formthereof that enhances an immune response to the tumor antigen.

In other illustrative embodiments, the N-terminal constant domaincomprises amino acids 88 to 97 of the P. aeruginosa flagellin (see,e.g., Verma et al., (2005) Infect. Immun. 73:8237-8246) or a homolog ormodified form thereof that enhances an immune response to the tumorantigen.

Regions of the flagellin protein involved in TLR5 signaling have beenidentified by Smith et al. (2003) Nat. Immunol. 4:1247-1253 (e.g., aminoacids 78-129, 135-173 and 394-444 of S. typhimurium flagellin orhomologs or modified forms thereof).

The flagellin N-terminal constant, C-terminal constant and hypervariableregions can be derived from flagellins from any suitable source, withsome or all of these regions being derived from the same organism orfrom different organisms. A number of flagellin genes have been clonedand sequenced (see, e.g., Kuwajima et al., (1986) J. Bact. 168:1479; Weiet al., (1985) J. Mol. Biol. 186:791-803; and Gill et al., (1983) J.Biol. Chem. 258:7395-7401). Non-limiting sources of flagellins includebut are not limited to S. enteritidis, S. typhimurium, S. dublin, H.pylori, V. cholera, S. marcesens, S. flexneri, S. enterica, T. pallidum,L. pneumophila, B. burgdorferei, C. difficile, A. tumefaciens, R.meliloti, B. clarridgeiae, R. lupine, P. mirabilis, B. subtilis, P.aeruginosa, and E. coli.

Non-limiting examples of fusion proteins of the invention are providedin the working Examples herein.

Optionally, the fusion protein can comprise any other peptide orprotein. For example, the fusion protein can further comprise one ormore antigens from other organisms. In representative embodiments, thefusion protein further comprises an immunomodulatory compound. Forexample, it is known in the art that immune responses can be enhanced byan immunomodulatory cytokine or chemokine (e.g., α-interferon,β-interferon, γ-interferon, co-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor,lymphotoxin, CCL25 [MECK], and CCL28 [TECH]). In particular embodiments,the fusion protein comprises γ-interferon and/or an IL-2-immunotoxin(e.g., an IL-2-diphtheria toxin).

The invention also provides a composition comprising a flagellinadjuvant and a tumor antigen. According to this embodiment, theflagellin adjuvant can be a full-length flagellin or can be a flagellinpeptide comprising the N-terminal constant and/or C-terminal constantregions as described in more detail above. Further, also as describedabove, the flagellin adjuvant can be coupled (i.e., fused) to a tumorantigen to form a fusion protein. The composition can comprise one ormore tumor antigens fused to the flagellin adjuvant and, optionally, oneor more tumor antigens present in the composition are not fused to theflagellin adjuvant. In other embodiments, the flagellin adjuvant is notcoupled to a tumor antigen.

Unless indicated otherwise, the fusion protein is administered per se asa protein (or a nucleic acid encoding the protein) and not as part oflive, killed, or recombinant bacterium- or virus-vectored vaccine.

4. Recombinant Nucleic Acids and Production of Fusion Proteins.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for cloning genes, amplifying and detectingnucleic acids, generating fusion constructs, expressing peptides in hostcells or organisms, and the like. Such techniques are known to thoseskilled in the art. See, e.g., Sambrook et al., “Molecular Cloning” ALaboratory Manual 2nd Ed. (Cold Spring Harbor, N. Y., 1989); F. M.Ausubel et al. Current Protocols in Molecular Biology (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York).

As used herein, “nucleic acid” encompasses both RNA and DNA, includingcDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA andchimeras of RNA and DNA. The nucleic acid may be double-stranded orsingle-stranded. The nucleic acid may be synthesized usingoligonucleotide analogs or derivatives (e.g., inosine orphosphorothioate nucleotides). Such oligonucleotides can be used, forexample, to prepare nucleic acids that have altered base-pairingabilities or increased resistance to nucleases.

The fusion protein of the invention can be produced in, and optionallypurified from, cultured cells or organisms expressing a heterologousnucleic acid encoding the fusion protein for a variety of purposes(e.g., to produce immunogenic compositions, as a diagnostic or researchreagent, and the like).

In some embodiments, the fusion protein can be collected and,optionally, purified from the host cell. For example, the fusion proteincan be collected from the conditioned medium. According to thisembodiment, it may be advantageous to express the fusion proteinoperably associated with a secretory signal sequence. Alternatively, thefusion protein can be isolated from the host cell (e.g., the host cellcan be lysed and the fusion protein isolated therefrom).

In other embodiments, the host cells are collected and the fusionprotein is not isolated therefrom.

Generally, the heterologous nucleic acid is incorporated into anexpression vector (viral or non-viral). Suitable expression vectorsinclude but are not limited to plasmids, bacteriophage, bacterialartificial chromosomes (bacs), yeast artificial chromosomes (yacs),cosmids, virus vectors, and the like. Expression vectors compatible withvarious host cells are well known in the art and contain suitableelements for transcription and translation of nucleic acids. Typically,an expression vector contains an “expression cassette,” which includes,in the 5′ to 3′ direction, a promoter, a coding sequence encoding thefusion protein operatively associated with the promoter, and,optionally, a termination sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system), yeast cells, mammalian cells, or plantcells. Examples of vectors for expression in yeast S. cerevisiae includepYepSecl (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Baculovirus vectors available for expression of nucleic acids to produceproteins in cultured insect cells (e.g., Sf9 cells) include the pAcseries (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow, V. A., and Summers, M. d. (1989) Virology 170:31-39).

Additionally, the expression vector will generally include expressioncontrol sequences (e.g., transcription/translation control signals andpolyadenylation signals), which are operably associated with the nucleicacid sequence encoding the fusion protein of the invention. It will beappreciated that a variety of promoter/enhancer elements can be useddepending on the level and tissue-specific expression desired. Thepromoter can be constitutive or inducible (e.g., the metallothioneinpromoter or a hormone inducible promoter), depending on the pattern ofexpression desired. The promoter can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thepromoter is not found in the wild-type host into which the promoter isintroduced. The promoter is chosen so that it will function in thetarget cell(s) of interest. Moreover, specific initiation signals aregenerally provided for efficient translation of inserted protein codingsequences. These translational control sequences, which can include theATG initiation codon and adjacent sequences, can be of a variety oforigins, both natural and synthetic. In embodiments of the inventionwherein the expression vector comprises two open reading frames to betranscribed, the open reading frames can be operatively associated withseparate promoters or with a single upstream promoter and one or moredownstream internal ribosome entry site (IRES) sequences (e.g., thepicornavirus EMC IRES sequence).

Examples of mammalian expression vectors include pCDM8 (Seed, (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand Simian Virus 40.

The invention further provides a host cell comprising (transiently orstably) a nucleic acid encoding a fusion protein of the invention.Suitable host cells are well-known in the art and include prokaryoticand eukaryotic cells. See e.g., Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Itis well-known that proteins can be expressed in bacterial cells such asE. coli, insect cells (e.g., the baculovirus expression system), yeastcells, plant cells or mammalian cells (e.g. human, rat, mouse, hamster,bovine, porcine, ovine, caprine, equine, feline, canine, lagomorph,simian and the like). The host cell can be a cultured cell such as acell of a primary or immortalized cell line. The host cell can be a cellin a microorganism, animal or plant being used essentially as abioreactor. In particular embodiments of the present invention, the hostcell is an insect cell that allows for replication of expressionvectors. For example, the host cell can be from Spodoptera frugiperda,such as the Sf9 or Sf21 cell lines, drosophila cell lines, or mosquitocell lines, e.g., Aedes albopictus derived cell lines. Use of insectcells for expression of heterologous proteins is well documented, as aremethods of introducing nucleic acids, such as vectors, e.g., insect-cellcompatible vectors (such as baculovirus vectors), into such cells andmethods of maintaining such cells in culture. See, for example, Methodsin Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly etal., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ.Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya etal., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J.Vir. 66:6922-30 (1992); Kimbauer et al., Vir. 219:37-44 (1996); Zhao etal., Vir. 272:382-93 (2000); and U.S. Pat. No. 6,204,059 to Samulski etal. In particular embodiments of the present invention, the insect cellis an Sf9 cell.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNAcomplexes, cell sonication, gene bombardment using high velocitymicroprojectiles, and viral-mediated transfection. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

In further embodiments of the present invention, the host cell can bestably transformed with the heterologous nucleic acid sequence encodingthe fusion protein. “Stable transformation” as used herein generallyrefers to the integration of the heterologous nucleic acid sequencesinto the genome of the host cell in contrast to “transienttransformation” wherein the heterologous nucleic acid sequenceintroduced into the host cell does not integrate into the genome of thehost cell. The term “stable transformant” can further refer to stablemaintenance of an episome (e.g. Epstein-Barr Virus (EBV)) in the cell.

When producing stably transformed cells, often only a small fraction ofcells (in particular, mammalian cells) integrate a foreign nucleic acidinto their genome. In order to identify and select these integrants, anucleic acid that encodes a selectable marker (e.g., resistance toantibiotics) can be introduced into the host cells along with thenucleic acid of interest. Preferred selectable markers include thosethat confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acids encoding a selectable marker can beintroduced into a host cell on the same vector as that comprising thenucleic acid of interest or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

The fusion protein can also be produced in a transgenic plant in whichthe isolated nucleic acid encoding the fusion protein is inserted intothe nuclear or plastidic genome. Plant transformation is known as theart. See, in general, Methods in Enzymology Vol. 153 (“Recombinant DNAPart D”) 1987, Wu and Grossman Eds., Academic Press and European PatentApplication EP 693554.

Foreign nucleic acids can be introduced into plant cells or protoplastsby several methods. For example, nucleic acid can be mechanicallytransferred by microinjection directly into plant cells by use ofmicropipettes. Foreign nucleic acid can also be transferred into a plantcell by using polyethylene glycol which forms a precipitation complexwith the genetic material that is taken up by the cell (Paszkowski etal. (1984) EMBO J. 3:2712-22). Foreign nucleic acid can be introducedinto a plant cell by electroporation (Fromm et al. (1985) Proc. Natl.Acad. Sci. USA 82:5824). In this technique, plant protoplasts areelectroporated in the presence of plasmids or nucleic acids containingthe relevant genetic construct. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and form a plant callus. Selection of the transformed plantcells comprising the foreign nucleic acid can be accomplished usingphenotypic markers.

Cauliflower mosaic virus (CaMV) can be used as a vector for introducingforeign nucleic acids into plant cells (Hohn et al. (1982) “MolecularBiology of Plant Tumors,” Academic Press, New York, pp. 549-560; Howell,U.S. Pat. No. 4,407,956). CaMV viral DNA genome is inserted into aparent bacterial plasmid creating a recombinant DNA molecule which canbe propagated in bacteria. The recombinant plasmid can be furthermodified by introduction of the desired DNA sequence. The modified viralportion of the recombinant plasmid is then excised from the parentbacterial plasmid, and used to inoculate the plant cells or plants.

High velocity ballistic penetration by small particles can be used tointroduce foreign nucleic acid into plant cells. Nucleic acid isdisposed within the matrix of small beads or particles, or on thesurface (Klein et al. (1987) Nature 327:70-73). Although typically onlya single introduction of a new nucleic acid segment is required, thismethod also provides for multiple introductions.

A nucleic acid can be introduced into a plant cell by infection of aplant cell, an explant, a meristem or a seed with Agrobacteriumtumefaciens transformed with the nucleic acid. Under appropriateconditions, the transformed plant cells are grown to form shoots, roots,and develop further into plants. The nucleic acids can be introducedinto plant cells, for example, by means of the Ti plasmid ofAgrobacterium tumefaciens. The Ti plasmid is transmitted to plant cellsupon infection by Agrobacterium tumefaciens, and is stably integratedinto the plant genome (Horsch et al. (1987) Science 227:1229-1231;Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803).

The fusion protein can be expressed from the nucleic acid operablyassociated with other peptides or proteins, for example, operablyassociated with purification signals (such as poly His) or as a chimerawith other proteins (e.g., a cytokine such as γ-interferon and/or anIL-2-immunotoxin such as an IL-2-diphtheria toxin).

5. Methods of Administering the Fusion Proteins, Flagellin Adjuvants,and Compositions of the Invention.

The present invention can be practiced for therapeutic and prophylacticpurposes, in accordance with known techniques (see, e.g., PCTApplication WO 2004/101737 to Pizzo et al.). The present invention canbe practiced prophylactically to prevent or reduce tumor formation. Inother embodiments, however, the methods of the invention are practicedto treat a subject that has already developed a tumor. Immunogeniccompositions for use in the inventive methods are described below.Boosting dosages can further be administered over a time course ofweeks, months or years. For existing tumors, initial high doses followedby boosting doses may be advantageous.

The invention can be practiced to treat subjects with existing tumors orto prevent or delay tumors from occurring. Further, the inventivemethods can be used to treat both a primary tumor and to preventmetastasis. In addition, the inventive methods can be advantageouslyemployed to reduce or prevent growth of metastatic nodules (e.g.,following surgical removal of a primary tumor). The methods of theinvention can also be prophylactic, e.g., to treat a subject believed atrisk for tumors.

By the term “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition and/or prevention or delay of the onset of a disease ordisorder. The term “treat,” “treats,” “treating,” or “treatment of” andthe like also include prophylactic treatment of the subject (e.g., toprevent the onset of infection or cancer). As used herein, the term“prevent,” “prevents,” or “prevention” (and grammatical equivalentsthereof) are not meant to imply complete abolition of disease andencompasses any type of prophylactic treatment that reduces theincidence of the condition, delays the onset and/or progression of thecondition, and/or reduces the symptoms associated with the condition.Thus, the term “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) refer to both prophylactic and therapeutic regimens.

As used herein, a method of “treating a tumor” in a subject includesboth therapeutic methods of treating existing tumors (including reducingthe incidence and/or the severity of metastasis) and prophylacticmethods of preventing or reducing tumor formation.

The terms “vaccination” or “immunization” are well-understood in theart, and are used interchangeably herein unless otherwise indicated. Forexample, the terms vaccination or immunization can be understood to be aprocess that increases an organism's immune response to antigen andtherefore to resist or overcome infection. In the case of the presentinvention, vaccination or immunization against the tumor antigenincreases the organism's immune response and resistance to tumors.

As used herein, a “treatment effective amount” is an amount that issufficient to treat (as defined herein) the subject.

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogens by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence and/or severity of disease.Alternatively, a protective immune response or protective immunity maybe useful in the therapeutic treatment of existing disease.

The present invention can be practiced for both medical and veterinarypurposes. Subjects to be treated by the methods of the invention includeboth avian and mammalian subjects, mammalian subjects including but notlimited to humans, non-human primates (e.g., monkeys, apes, baboons, andchimpanzees), dogs, cats, rabbits, goats, horses, pigs, cattle, sheep,and the like (including both male and female subjects and subjects ofall ages including infant, juvenile, adolescent and adult subjects).Subjects may be treated for any purpose, such as for eliciting aprotective immune response; for eliciting the production of antibodiesin that subject (typically an animal subject) which antibodies can becollected and used for other purposes such as diagnostic purposes oradministering to other subjects to produce passive immunity therein,etc.

In particular embodiments, the subject is an animal tumor model. Inother embodiments, the subject has a tumor and/or is a subject believedat risk for tumors.

Optionally, the subject can be a subject “in need of” the fusionproteins, compositions, pharmaceutical formulations and methods of theinvention (e.g., to protect against cancer).

In some embodiments the subjects are aged subjects, e.g., human subjects50 or 60 years old or more, where other adjuvants such as alum aregenerally less effective.

Accordingly, in particular embodiments, the invention provides a methodof inducing an immune response against a tumor antigen in a subject(such as a mammalian subject, e.g., human or primate), the methodcomprising administering a fusion protein of the invention or apharmaceutical composition thereof to the subject in an immunogenicallyeffective amount. In representative embodiments, the method is practicedas a method of treating a tumor in a subject (such as a mammaliansubject, e.g., human or primate), the method comprising administering afusion protein of the invention or a pharmaceutical composition thereofto the subject in a treatment effective amount.

The invention further provides a method of inducing an immune responseto a tumor antigen in a subject (such as a mammalian subject, e.g.,human or primate), the method comprising administering a flagellinadjuvant and a tumor antigen, or a pharmaceutical composition(s)thereof, to the subject in an immunogenically effective amount. Inrepresentative embodiments, the method is practiced as a method oftreating a tumor in a subject (such as a mammalian subject, e.g., humanor primate), the method comprising administering a flagellin adjuvantand a tumor antigen, or a pharmaceutical composition(s) thereof, to thesubject in a treatment effective amount. The flagellin adjuvant andtumor antigen can be administered in the same or separate compositions.If administered as separate compositions, they can optionally beadministered concurrently.

Administration can be by any route known in the art. As non-limitingexamples, the route of administration can be by inhalation (e.g., oraland/or nasal inhalation), oral, buccal (e.g., sublingual), rectal,vaginal, topical (including administration to the airways), intraocular,transdermal, by parenteral (e.g., intramuscular [includingadministration to skeletal, cardiac and/or diaphragm muscle],intravenous, subcutaneous, intradermal, intrapleural, intracerebral andintra-arterial, and intrathecal) routes, as well as direct tissue ororgan injection, or by administration to the central nervous system(e.g., stereotactic administration to the brain).

In particular embodiments, administration is to a mucosal surface, e.g.,by intranasal, inhalation, intra-tracheal, oral, rectal or vaginaladministration, and the like. In general, mucosal administration refersto delivery to a mucosal surface such as a surface of the respiratorytract, gastrointestinal tract, urinary tract, reproductive tract, etc.

Methods of administration to the respiratory tract include but are notlimited to transmucosal, intranasal, inhalation or intratrachealadministration or administration to the lungs. Other methods of mucosaladministration include oral, buccal (e.g., sub-lingual), intra-tracheal,rectal, vaginal and intra-ocular administration.

In some embodiments of the invention, administration is in or near thesite of a tumor. In other embodiments, administration is directly intothe lymphatic system (e.g., in or near a lymph node).

The protein(s) of the invention can be delivered per se or by deliveringa nucleic acid intermediate that encodes the protein(s) and is expressedin the subject to produce the protein(s), such as described in U.S. Pat.No. 5,589,466 to Felgner et al.

Immunomodulatory compounds, such as immunomodulatory chemokines andcytokines (preferably, CTL inductive cytokines) can be co-administeredto a subject. Cytokines may be administered by any method known in theart. Exogenous cytokines may be administered to the subject, oralternatively, a nucleotide sequence encoding a cytokine may bedelivered to the subject using a suitable vector, and the cytokineproduced in vivo. In particular embodiments, the cytokine is provided asfusion protein with the flagellin adjuvant and/or tumor antigen. Forexample, a fusion protein comprising a flagellin adjuvant, a tumorantigen, and an immunomodulatory cytokine (e.g., interferon-γ or anIL-2-immunotoxin) can be administered. Alternatively, a fusion proteincomprising the cytokine and the tumor antigen or the flagellin adjuvantcan be administered.

In addition to their use for prophylactic or therapeutic purposes, thefusion proteins and compositions of the present invention can beadministered to subjects for the purpose of producing antibodies to atumor antigen, which antibodies are in turn useful for diagnostic ortherapeutic/prophylactic purposes in human and animal subjects.

6. Pharmaceutical Compositions.

The invention further provides pharmaceutical compositions (e.g.,immunogenic compositions) comprising a fusion protein of the inventionin a pharmaceutically acceptable carrier. In particular embodiments, thepharmaceutical composition is formulated for mucosal delivery. By“pharmaceutically acceptable” it is meant a material that is not toxicor otherwise undesirable.

In representative embodiments, the fusion protein is present in thepharmaceutical composition in an “immunogenically effective” amount. An“immunogenically effective amount” is an amount that is sufficient toevoke an active immune response (i.e., cellular and/or humoral) in thesubject to which the pharmaceutical composition is administered.Optionally, the dosage is sufficient to produce a protective immuneresponse (prophylactic or therapeutic after onset of infection). Thedegree of protection conferred need not be complete or permanent, aslong as the benefits of administering the pharmaceutical compositionoutweigh any disadvantages thereof. Immunogenically effective amountsdepend on the protein, the manner of administration, the stage andseverity of the disease being treated, the weight and general state ofhealth of the subject, and the judgment of the prescribing physician.

Dosages of pharmaceutically active compounds can be determined bymethods known in the art, see, e.g., Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). In particular embodiments, thedosage of the fusion proteins of the present invention ranges from about0.1, 0.5, 1, 10, 25, 250, 100, 150 or 250 μg to about 300, 500, 1000,2500, 5000 or 10,000 μg of fusion protein for a typical (e.g. 70 kg)subject. In particular embodiments, dosages are in the range of about 50to 2000 μg, about 100 to 1500 μg, or about 250 to 1000 μg for a typicalsubject. The initial dose can be followed by boosting dosages overweeks, months or years of from about 1 μg to 250, 500 or 1000 μgdepending on the subject's response to the initial dosage.

The invention also provides a pharmaceutical composition comprising: (a)a flagellin adjuvant; and (b) a tumor antigen. In particularembodiments, the pharmaceutical composition is formulated for mucosaladministration. Optionally, the tumor antigen is coupled to theflagellin adjuvant, i.e., is in the form of a fusion protein with theflagellin antigen. According to this embodiment, the composition canfurther comprise one or more additional tumor antigens that are notcoupled to the flagellin adjuvant (i.e., is not part of a fusion proteinwith the flagellin adjuvant).

Optionally, the tumor antigen is present in an immunogenically effectiveamount, as defined herein. Further, in some embodiments, the flagellinadjuvant is present in an “adjuvant effective amount.” An “adjuvanteffective amount” is an amount of the flagellin adjuvant that issufficient to enhance or stimulate the active immune response (cellularand/or humoral) mounted by the host against the tumor antigen,optionally an active mucosal immune response. In particular embodiments,the active immune response (e.g., a mucosal immune response) by the hostis enhanced by at least about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60,75, 100, 150, 500, 1000-fold or more. In other embodiments, an “adjuvanteffective amount” is an amount of the flagellin adjuvant that reducesthe amount of antigen required to achieve a specified level of immunity(cellular and/or humoral), optionally mucosal immunity, for example, areduction of at least about 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%, 90%,95%, 98% or more in the amount of antigen. As a further option, an“adjuvant effective amount” can refer to an amount of the flagellinadjuvant that accelerates the induction of the immune response in thehost and/or reduces the need for booster immunizations to achieveprotection. As yet another alternative, an “adjuvant effective amount”can be an amount that prolongs the time period over which an immuneresponse, optionally protective immune response, is sustained (e.g., byat least about a 2-fold, 3-fold, 5-fold, 10-fold, 20-fold longer timeperiod or more).

Dosages of the flagellin adjuvant and tumor antigen (if not in the formof a fusion protein) can be determined by those skilled in the art. Inparticular embodiments, dosages of the flagellin adjuvant are in therange from about 0.1, 0.5, 1, 10, 25, 50, 100 or 150 μg to about 200,250, 300, 500, 1000, or 2500 μg for a typical (e.g., 70 kg) subject. Inparticular embodiments, dosages are from about 10 to 1000 μg, or fromabout 50 to 500 μg, or from about 150 to 300 μg for a typical subject.Suitable dosages of the tumor antigen can range from about 0.1, 0.5, 1,10, 25, 50, 100 or 150 μg to about 200, 300, 500, 1000, 1500, 2000,2500, 5000 or 10,000 μg for a typical (e.g. 70 kg) subject. Inparticular embodiments, the dosage of the tumor antigen is from about 50to 2000 μg, from about 150 to about 1500 μg, or from about 300 to about1000 μg for a typical subject. The initial dose can be followed byboosting dosages over weeks, months or years of from about 1 μg to about1000 μg depending on the subject's response to the initial dosage.

The pharmaceutical compositions of the invention can optionally compriseother medicinal agents, pharmaceutical agents, stabilizing agents,buffers, carriers, diluents, salts, tonicity adjusting agents, wettingagents, and the like, for example, sodium acetate, sodium lactate,sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

For injection, the carrier will typically be a liquid. For other methodsof administration, the carrier may be either solid or liquid. Forinhalation administration, the carrier will be respirable, and istypically in a solid or liquid particulate form.

While adjuvants beyond flagellin are generally not required, thecomposition can optionally comprise an additional adjuvant, such ascomplete or incomplete Freund's adjuvant, aluminum phosphate, aluminumhydroxide, alum, cytokines, TLR ligands, and the like.

The concentration of the proteins in the pharmaceutical compositions canvary widely, e.g., from less than about 0.01% or 0.1% up to at leastabout 2% to as much as 20% to 50% or more by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

The proteins can be formulated for administration in a pharmaceuticalcarrier in accordance with known techniques. See, e.g., Remington, TheScience And Practice of Pharmacy (9^(th) Ed. 1995). In the manufactureof a pharmaceutical composition according to the invention, theprotein(s) (including physiologically acceptable salts thereof) istypically admixed with, inter alia, an acceptable carrier. The carriercan be a solid or a liquid, or both, and is optionally formulated withthe compound as a unit-dose formulation, for example, a tablet. Avariety of pharmaceutically acceptable aqueous carriers can be used,e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid,pyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.), andthe like. These compositions can be sterilized by conventionaltechniques. One or more proteins can be incorporated in the formulationsof the invention, which can be prepared by any of the well-knowntechniques of pharmacy.

The pharmaceutical compositions can be packaged for use as is, orlyophilized, the lyophilized preparation generally being combined with asterile aqueous solution prior to administration. The compositions canfurther be packaged in unit/dose or multi-dose containers, for example,in sealed ampoules and vials.

The pharmaceutical compositions can be formulated for administration byany method known in the art according to conventional techniques ofpharmacy. For example, the compositions can be formulated to beadministered intranasally, by inhalation (e.g., oral inhalation),orally, buccally (e.g., sublingually), rectally, vaginally, topically,intrathecally, intraocularly, transdermally, by parenteraladministration (e.g., intramuscular [including administration toskeletal, cardiac and/or diaphragm muscle], intravenous, subcutaneous,intradermal, intrapleural, intracerebral and intra-arterial,intrathecal), topically (e.g., to both skin and mucosal surfaces,including airway surfaces), as well as direct tissue or organ injection,and by administration to the central nervous system (e.g., stereotacticadministration to the brain).

In some embodiments of the invention, the pharmaceutical composition isformulated for administration in or near the site of a tumor. In otherembodiments, the pharmaceutical composition is formulated foradministration directly into the lymphatic system (e.g., in or near alymph node).

In particular embodiments, the pharmaceutical composition isadministered to a mucosal surface, e.g., by intranasal, inhalation,intratracheal, oral, rectal or vaginal administration, and the like.

For intranasal or inhalation administration, the pharmaceuticalcomposition can be formulated as an aerosol (this term including bothliquid and dry powder aerosols). For example, the pharmaceuticalcomposition can be provided in a finely divided form along with asurfactant and propellant. Typical percentages of the composition are0.01-20% by weight, preferably 1-10%. The surfactant is generallynontoxic and soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1-20% by weight of the composition, preferably 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, if desired, as with lecithin for intranasal delivery. Aerosolsof liquid particles can be produced by any suitable means, such as witha pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as isknown to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.Aerosols of solid particles can likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art. Intranasal administration can also be by dropletadministration to a nasal surface.

Injectable formulations can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one can administer the pharmaceutical composition in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which ispharmaceutically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortables, as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilemulsion. Oral delivery can be performed by complexing a compound(s) ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriersinclude plastic capsules or tablets, as known in the art. Suchformulations are prepared by any suitable method of pharmacy, whichincludes the step of bringing into association the compound(s) and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the pharmaceutical compositions are preparedby uniformly and intimately admixing the compound(s) with a liquid orfinely divided solid carrier, or both, and then, if necessary, shapingthe resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the compound(s),optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the compound(s) in a flavoredbase, usually sucrose and acacia or tragacanth; and pastilles comprisingthe compound(s) in an inert base such as gelatin and glycerin or sucroseand acacia.

Pharmaceutical compositions of this invention suitable for parenteraladministration can comprise sterile aqueous and non-aqueous injectionsolutions of the compounds of this invention, which preparations arepreferably isotonic with the blood of the intended recipient. Thesepreparations can contain anti-oxidants, buffers, bacteriostats andsolutes, which render the composition isotonic with the blood of theintended recipient. Aqueous and non-aqueous sterile suspensions,solutions and emulsions can include suspending agents and thickeningagents. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

Pharmaceutical compositions suitable for rectal administration arepreferably presented as unit dose suppositories. These can be preparedby admixing the compound(s) with one or more conventional solidcarriers, such as for example, cocoa butter and then shaping theresulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers that can be usedinclude, but are not limited to, petroleum jelly, lanoline, polyethyleneglycols, alcohols, transdermal enhancers, and combinations of two ormore thereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharmaceutical Research3:318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the compound(s). Suitable formulations can comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and can contain from0.1 to 0.2M active ingredient.

Further, the compound(s) can be formulated as a liposomal formulations.The technology for forming liposomal suspensions is well known in theart. When the compound(s) or salt thereof is an aqueous-soluble salt,using conventional liposome technology, the same can be incorporatedinto lipid vesicles. In such an instance, due to the water solubility ofthe compound(s) or salt, the compound(s) or salt will be substantiallyentrained within the hydrophilic center or core of the liposomes. Thelipid layer employed can be of any conventional composition and caneither contain cholesterol or can be cholesterol-free. When thecompound(s) or salt of interest is water-insoluble, again employingconventional liposome formation technology, the salt can besubstantially entrained within the hydrophobic lipid bilayer which formsthe structure of the liposome. In either instance, the liposomes whichare produced can be reduced in size, as through the use of standardsonication and homogenization techniques.

The liposomal formulations can be lyophilized to produce a lyophilizatewhich can be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention. Abbreviations used herein are as follows: GM-CSF,granulocyte-macrophage colony stimulating factor; IL, interleukin; i.n.,intranasal; i.t., intra-tracheal; NK, natural killer cell; NO, nitricoxide; s.c., subcutaneous; TLR, toll like receptor; TGF-β; transforminggrowth factor beta.

Example 1 Cancer Antigens and Vaccines Thereof

Effect of flagellin on innate immunity in the mouse lung. Non-surgicalintratracheal (i.t.) instillation of 1 μg of flagellin is sufficient toinduce maximal production of TNFα after approximately 4 hr (FIG. 1). By12-24 hr, cytokine levels in broncheoalveolar lavage fluid return tobaseline levels. Note that a mutant flagellin that does not bind to TLR5and thus lacks signaling activity, did not induce cytokine production.In addition to TNFα, several other cytokines including IL-6, G-CSF, andthe chemokines, MIP-2 and KC were induced to relatively high levels. Theincrease in cytokine expression is followed by the transientinfiltration of neutrophils (maximal at 12-24 hr). It is important toemphasize that the innate immune response initiated by flagellin doesnot result in severe tissue damaging inflammation. The inducedinflammatory response is relatively moderate and acute in nature. Thesefindings, in conjunction with those of other investigators, establishthe in vivo potency of flagellin as an activator of innate immunity.

Adjuvant effect of flagellin in the mouse lung. In addition to analyzingthe innate response induced by flagellin, the effects of flagellin onthe antibody response were examined. BALB/c mice were immunized with 10μg F1 antigen with 1 μg flagellin or an inactive flagellin mutantprotein by nonsurgical intra-tracheal (i.t.) or intra-nasal (i.n.)instillation. Four weeks later, the mice were boosted with the sameregimens and then serum levels of anti-F1 IgG were determined two weekslater. Flagellin, but not the inactive mutant, exhibited extraordinarilypotent adjuvant activity as measured by serum anti-F1 IgG levels. Inanimals immunized i.t., the antibody titers ranged from 20,000 togreater than 100,000, and from 90,000 to greater than 300,000 in thecase of animals immunized i.n. The results for i.n. immunization arepresented in Table 1. Analysis of the cytokine requirements for thisresponse using knockout mice indicate that neither IL-6, IL-12, norTNF-α are required for the adjuvant effect of flagellin (FIG. 5).Furthermore, the maximal adjuvant effect of flagellin is achieved with adose of flagellin (1 μg) that is 5-10× less than required for themaximal induction of the innate immune response in the respiratory tractof mice. Thus maximal adjuvant activity is achieved with a dose offlagellin that produces limited inflammation.

TABLE 1 I.n. immunization with Y. pestis F1 antigen and wild-typeflagellin results in a strong anti-F1 IgG response. Mice were given twoimmunizations with 10 μg F1 antigen and 1 μg wild-type or mutantflagellin. Serum anti-F1 IgG titers were measured by ELISA. FlagellinTotal IgG Titer Inactive mutant (7 mice) <100 wild-type mouse 1 >300,000mouse 2 >300,000 mouse 3 >300,000 mouse 4 90,000 mouse 5 >300,000 mouse6 300,000

Example 2 Flagellin as an Adjuvant to Produce an Antigen-Specific BreastCancer Response

Treatment of breast tumor in vivo. BALB/c mice were immunized with theFra-1 antigen, an antigen that is over-expressed by many breast tumors,and either flagellin or an inactive form of flagellin. The mice wereboosted once prior to subcutaneous injection of D2F2 cells, a breastcancer tumor. The mice were monitored for the growth of tumors and thetumor volumes were determined. Data are shown in FIG. 2. The opencircles represent the mice given the Fra-1 antigen and the inactive formof flagellin. The closed circles represent the mice given Fra-1 antigenand the active form of flagellin. As can readily be seen,flagellin+Fra-1 antigen had a significant effect on the growth of thetumor.

Evaluation of whether flagellin can promote a protective immune responseagainst D2F2 breast cancer cells in a mouse model. The ability offlagellin to promote a protective state of immunity against D2F2 breastcancer cells is evaluated using as an antigen, Fra-1, a protein that isover-expressed by these cancer cells These experiments involveimmunization and then challenge with D2F2 cells as well as challengefollowed by immunization. In addition, the nature of the inducedeffectors-antibodies vs. cytolytic T cells and/or NK cells isdetermined.

A. Preparation of purified recombinant Fra-1 antigen. The pET22expression vector encoding the cDNA encoding murine Fra-1 (kindlyprovided by Dr. Rong Xiang, The Scripps Research Institute) is used toexpress the Fra-1 protein in Rosetta-gami-pLysS bacteria. The protein ispurified using the Talon affinity resin (via recognition of the His-tagon the Fra-1) and contaminating endotoxin is removed by passage of thepurified protein over a Detoxi-gel column (McDermott et al. (2000)Infect. Immun. 68:5525-5529). Using this procedure, mg amounts ofseveral proteins have been obtained previously (e.g., flagellin, F1antigen, and IRAK-4 [a eukaryotic protein]).

B. Evaluation of whether flagellin promotes protective immunity againstD2F2 breast cancer cells in BALB/c mice immunized with Fra-1 antigen andflagellin. Groups of 7 mice are immunized with 10 μg Fra-1 protein withor without 1 μg flagellin or mutant flagellin 229 intra-peritoneally(i.p.) or intramuscularly (i.m.). Two weeks later, the mice are boostedand then challenged with 1×10⁶ D2F2 cells (given subcutaneously (s.c.)).Tumor mass and the survival of the mice is followed over a period ofapproximately 3 months. In the absence of immunity, approximately 50% ofthe mice succumb within 4 weeks after challenge. The s.c. tumors aremeasured for width and length so that tumor volume can be calculated. Ifa significant effect of flagellin is observed, the dose of flagellinthat produces the maximal response is determined. Once the optimal doseof flagellin is established, it is determined if flagellin can promotethe clearance of an existing tumor. Groups of 7 mice are challenged withD2F2 cells by s.c. injection and then immunized with Fra-1 with orwithout flagellin at the time of challenge or 1, 3, 7, and 21 dayslater. In each case, the mice are boosted after 2 weeks. Tumor mass andsurvival of the mice are determined.

C. Identification of the potential effectors in the flagellin-promotedresponse against Fra-1-expressing D2F2 cells. To identify the inducedeffectors in the flagellin-promoted response, the level of circulatinganti-Fra-1 antibodies as well as the relative numbers of CD8+ cytolyticT cells (CTL) and NK cells are determined. BALB/c mice are immunizedwith Fra-1 and flagellin or mutant flagellin according to the protocolpresented above.

1. Anti-Fra-1 antibodies. Two weeks after challenge with D2F2 cells, themice are euthanized and serum samples are taken for analysis ofcirculating anti-Fra-1 antibodies by ELISA. Using appropriateanti-isotypic antibodies, the levels of circulating total IgG and IgM aswell as IgG1 and IgG2a sub-isotypes are assessed.

2. Anti-Fra-1 specific CD8+ CTL. Anti-Fra-1 specific CD8+ CTL aredetermined by isolating splenocytes and assessing their cytolyticactivity in a standard ⁵¹Cr-release assay. The lysis mediated by CD8+CTL should be blocked by an anti-MHC class I antibody. Therefore, cellsamples are analyzed in the presence and absence of this antibody. Byvarying the ratio of effectors-to-targets, a relative estimate of theincrease in CD8+ CTL in mice immunized with Fra-1 and flagellin ormutant flagellin is obtained. The numbers of activated CD8+ CTL can alsobe determined using an ELISPOT assay for interferon-γ (IFN-γ)production. Using anti-CD8 MACS MicroBeads (Miltenyl Biotech), CD8+splenocytes from immunized mice are isolated and the cells incubated for24 h with irradiated D2F2 cells. The cells are analyzed by ELISPOT assayfor IFN-γ production. If sufficient numbers of cells are present,intracellular cytokine staining (ICS) is used as a second method.

3. NK cells. The relative numbers of activated NK cells are assessed ina ⁵¹Cr-release assay using YAC-1 cells as targets. Splenocytes areobtained from immunized and D2F2 challenged mice and assessed forcytolytic activity against YAC-1 cells. Since the expression of DX5 isassociated with NK cells, the expression of this marker by flowcytometry is measured using a commercially available PE-labeledantibody. A stimulatory effect of flagellin on the expansion of NK cellsshould be associated with an increase in DX5 expression.

The efficacy of a recombinant flagellin protein containing Fra-1epitopes in the protective response against D2F2 breast cancer cells. Itis further determined if flagellin can serve as a vaccine vector andadjuvant. If this is the case, recombinant flagellin proteins encoding anumber of epitopes from different tumor antigens are generated.

The available evidence indicates that human breast cancers exhibitsubstantial heterogeneity in the tumor-specific antigens that theyexpress. For example, MAGE-3 is expressed in approximately 14% of breastcancers, whereas Her2/neu is expressed in 40%, NY-BR-62 in 60%, andNY-BR-85 in approximately 90% (Scanlan and Jäger (2001) Breast CancerRes. 3:95-98). It is determined if a recombinant flagellin proteincontaining full-length Fra-1 antigen can function in the same manner asthe two proteins.

If this is the case, then the epitopes within Fra-1 that are involved ininducing a protective response are mapped. It would be a straightforwardeffort to do the same thing with other major breast cancer antigentargets. A flagellin protein expressing epitopes from a range of targetantigens can be generated. Alternatively, a cocktail of flagellinproteins can be used—each flagellin expressing a subset of targetepitopes. Furthermore, by introducing foreign epitopes are introducedinto the hypervariable region of the protein—a region that is notinvolved in the interaction of flagellin with TLR5 (Donnelly and Steiner(2002) J. Biol. Chem. 277:40456-40461; Smith et al. (2003) Nat. Immunol.4:1247-1253; Murthy et al. (2004) J. Biol. Chem. 279:5667-5675), it isunlikely that the biologic activities of flagellin are affected in anysignificant manner.

Generation of a flagellin/Fra-1 chimera. The full sequence for Fra-1 isinserted into the hypervariable region of flagellin and the resultantconstruct is cloned into the pET22a expression vector and the protein isexpressed in Rosetta-gami-pLysS bacteria. Endotoxin is removed using aDetoxi-gel column. The biologic activity of the chimeric protein isassessed using TNFα production by RAW264.7 cells. The chimera istitrated and its potency compared with wild-type flagellin.

Mapping of Fra-1 epitopes required for protection against D2F2 cells.The ability of the chimera to protect BALB/c mice against a challengewith D2F2 cells is evaluated. If the chimera induces protectiveimmunity, overlapping truncations of the Fra-1 sequence are generatedand tested for efficacy in the context of the flagellin. To reduce thenumber of constructs, three overlapping fragments covering the entiresequence of Fra-1 are prepared. If one of these truncations is active,additional truncations are generated to define the minimal sequence(s)that is required for protection. If none of the three truncations isfunctional by itself, these are tested in pairs to determine if therequired sequences are in different parts of the protein. Using thisapproach, the minimal required sequences can be defined.

Example 3 Flagellin is an Effective Mucosal Adjuvant for ImmunizationAgainst Lethal Respiratory Challenge with Yersinia pestis Methods:

Plasmids and cell culture. The coding sequence for the F1 antigen ofYersinia pestis, caf1, (plasmid containing the entire caf operon kindlyprovided by Dr. J. B. Bliska, State University of New York, Stony Brook)was subcloned into the NdeI and XhoI sites of the pET29a expressionvector from Novagen (EMD Biosciences, Inc., Madison, Wis.). Therecombinant F1/V fusion construct (Heat et al. (1998) Vaccine16:1131-1137) (provided by Drs. G. Andrews and P. Worsham, USAMRIID) wassequenced and subcloned into pET16b. Sequencing revealed the absence of21 amino acids corresponding to the signal sequence of F1.

Reagents and antibodies. Purified, recombinant His-tagged flagellin fromSalmonella enteritidis was prepared as described previously (Honko andMizel (2004) Infect. Immun. 72:6676-6679; McDermott et al. (2000)Infect. Immun. 68:5525-5529). The 229 mutant flagellin, as well as theF1 and F1/V antigens were purified in an identical manner. Endotoxinlevels were ≦1 pg/μg as detected by the QCL-1000® Chromogenic LAL TestKit from the Cambrex Corporation (East Rutherford, N.J.). TNF-α wasdetected using the BD OptEIA ELISA kit (mono/mono) per the manufacturersinstructions (BD Biosciences). An anti-F1 mouse monoclonal IgG1, cloneYPF19 obtained from Research Diagnostics, Inc. (Flanders, N.J.), wasused as a control in the anti-F1 ELISA. Goat anti-mouse IgG-HRP waspurchased from SouthernBiotech (Birmingham, Ala.). Goat anti-monkeyIgG-HRP was purchased from Research Diagnostics, Inc. (Flanders, N.J.).

Mice. Female BALB/cAnNCr mice were purchased from the Frederick CancerResearch and Development Center (Frederick, Md.). Female IL-6−/− mice(B6; 129S2-116/J), TNFR1 mice (tm1Kopf−/− B6; 129S-Tnfrsf1a Tnfrsf1b/J),IFNγ (tm1Imx tm1Imx −/− B6.129S7-Ifngtm1Ts/J) and control mice(C57BL/6J, B6; 129SF2/J and 129/SvJ) were purchased from The JacksonLaboratory (Bar Harbor, Me.). IFNα/βR−/− mice were provided by Dr. C.Schindler, Columbia University, New York (Müller et al. (1994) Science264:1918-1921). Mice were maintained in a specific-pathogen freefacility and all research complied with federal and institutionalguidelines set forth by the Wake Forest University Animal Care and UseCommittee.

Nonsurgical intratracheal and intranasal immunization of mice. Forintratracheal immunization, mice were anesthetized with Avertin(2,2,2-tribromoethanol, Sigma; tert-amyl alcohol, Fisher) byintraperitoneal injection and suspended from a length of wire by theirfront incisors. Using a sterile gel-loading tip inserted gently into thetrachea, 10 μg F1 antigen and the indicated amount of flagellin or theflagellin mutant 229 was administered in a total of 50 μL pyrogen-freePBS. For intranasal immunization, small volumes (9-12 μL total)containing antigen and adjuvant in PBS were administered to the nostrilsof anesthetized mice. Mice were boosted at 4 weeks and plasma collected2-3 weeks post-boost for analysis of antibody titers.

Immunization of monkeys. Fifteen healthy adult female cynomolgus monkeys(Macaca fascicularis) were maintained in accordance with federal andinstitutional guidelines set forth by the Wake Forest University AnimalCare and Use Committee. Animals were anesthetized with 7-10 mg/kgketamine intramuscularly for immunizations and blood collection. Forintranasal immunization, 150 μg F1/V fusion protein and 50 μg flagellinwere delivered dropwise (100 μL/nostril) to animals in a recumbentposition. Intramuscular immunizations were administered into thequadriceps in a volume of 1 mL. Control animals received PBSintranasally and intramuscularly.

Analysis of plasma antibody titers by enzyme-linked immunosorbant assay(ELISA). Heparinized tubes (StatSpin; Fisher Scientific) or BDVacutainer PST tubes were used for plasma collection. Plasma was thenaliquoted and frozen at −70° C. until analysis. ELISA plates were coatedwith 100 μL of antigen at 10 μg/mL in sterile PBS overnight at 4° C. andblocked with 10% FCS in PBS. Duplicate or triplicate plasma dilutionswere added and the plate was incubated overnight at 4° C., followed bysecondary anti-Ig antibodies for 2 h at room temperature. Peroxidaseactivity was detected with 3,3′,5,5′-Tetramethylbenzidine (TMB) LiquidSubstrate System (Sigma-Aldrich) and stopped with 2M H₂SO₄. End-pointdilution titers were defined as the inverse of the highest dilution thatresulted in an absorbance value (OD₄₅₀)>0.1 over that of naive plasma.

Respiratory challenge with Yersinia pestis CO92. The Centers for DiseaseControl (CDC) Division of Vector-Borne Infectious Diseases (FortCollins, Colo.) provided a stock culture of Yersinia pestis CO92 biovarorientalis, a strain isolated from a fatal case of human primarypneumonic plague (Doll et al. (1994) Am. J. Trop. Med. Hyg. 51:109-114).Heart infusion broth was inoculated with a single colony from asubculture plate and grown at 28° C. to an approximate density of 1×10⁹colony forming units (cfu)/mL. Mice were challenged intranasally with 10μL of culture diluted in PBS to ˜1.8×10⁷ cfu/mL, a dose equivalent to150× lethal dose 50% (LD50) of 1.2×10⁴ cfu (data not shown). Actualcfu/mL values were determined by plating serial dilutions onto tryptoseblood agar plates. Survival was monitored for 14-30 days post challenge.All experiments were conducted according to CDC approved standardoperating procedures for the BSL3 and ABSL3 facility at the InfectiousDisease Unit at Virginia Tech (CDC approval #C20031120-0016).

Statistical analysis. Data are represented as individual values withmeans and standard error. The F test for equality of variance andStudent's one-sided t-test were used to assign statistical significanceat p<0.05 or p<0.01. SigmaStat3.1 (Systat Software, Inc., Richmond,Calif.) was used to determine LD₅₀ values by nonlinear regression.

Results:

Immunization with flagellin promotes a potent adaptive response to theF1 antigen of Yersinia pestis. To determine the ability of flagellin topromote a humoral immune response against the F1 antigen of Y. pestis,BALB/c mice were immunized with 10 μg F1 antigen and 1 μg recombinantflagellin (FliC) intratracheally (i.t.) or intranasally (i.n.). Controlanimals were immunized with F1 antigen in PBS, or F1 and a mutant formof flagellin, designated 229. Four weeks later, mice were boosted in anidentical manner and plasma was collected for analysis of circulatingantibody titers at various times post-boost. No F1-specific IgG wasdetected in the groups of control mice. However, a vaccine containing F1and flagellin stimulated a dramatic increase in total IgG titers (FIG.3, panel a), with high levels of F1-specific IgG1 and IgG2a. The meanratio of IgG1 to IgG2a (indicated within the bars) ranged from 30-170when flagellin and F1 were administered i.t. and was approximately 3when given i.n. Although i.n. and i.t. immunization resulted in mixed Thresponses, the bias towards an apparent Th2 response was more evidentfollowing intratracheal immunization. It is important to note thatflagellin did not promote significant F1-specific IgE production. Miceimmunized with F1 and flagellin exhibited sustained titers of anti-F1IgG after two immunizations (FIG. 3, panel b). A tertiary immunizationat 16 weeks improved the antibody response in the two mice that hadlower initial titers.

In a previous study, it was demonstrated that the innate response toflagellin in the lung was maximal with doses of flagellin in the rangeof 5-15 μg (Honko and Mizel (2004) Infect. Immun. 72:6676-6679). Todetermine if there was a linear relationship between the magnitude ofthe cytokine response and the antibody response promoted by flagellin,BALB/c mice were immunized with F1 antigen and 1 μg, 5 μg and 15 μg offlagellin (FIG. 3, panel c). Anti-F1 IgG titers were not significantlydifferent at these doses, indicating that the adjuvant effect is maximalat 1 μg flagellin. However, we noticed a trend towards a greaterIgG1/IgG2a ratio with increasing doses of flagellin. Thus it appearsthat a maximal adaptive response may not require a maximal innateresponse.

Pre-existing immunity to flagellin is an obvious concern whenconsidering its use as an adjuvant. Therefore, we evaluated theeffectiveness of immunization with flagellin and F1 in the presence ofhigh titers of anti-flagellin antibodies. Female BALB/c mice wereimmunized and boosted with 5 μg flagellin i.n. and anti-flagellinantibody titers were determined. Flagellin-specific IgG titers werebelow the level of detection prior to immunization, and increasedsignificantly with a mean anti-FliC IgG titer of 8.5×10⁵. These micewere then immunized and boosted with 10 μg F1 and 1 μg FliC i.n. Twoweeks post-boost, anti-F1 IgG titers were similar between naive andFliC-immune mice (FIG. 3, panel d), indicating that circulatinganti-flagellin antibodies did not positively or negatively alter theresponse to flagellin. Our results, support the conclusion thatflagellin is an effective adjuvant in the presence of prior immunity toflagellin.

The adjuvant effect of flagellin stimulates antigen-specific responsesand is dependent on T lymphocytes. The development of immunologicalmemory is an essential feature of an effective vaccine, preparing theimmune system to respond rapidly following subsequent exposure to theantigen during infection. To evaluate the requirement for flagellinstimulation in the secondary immunization, four groups of BALB/c micewere immunized with F1 antigen and flagellin and subsequently boostedwith PBS, flagellin alone, F1 antigen alone, or flagellin and F1 (FIG.4, panel a). F1-specific IgG titers in mice boosted with PBS orflagellin alone remained between 500-1100, values that are typical forpost-primary responses. However, mice that received F1 antigen alone inthe secondary immunization had dramatically increased anti-F1 IgGtiters. Although flagellin was not required in the boost, there was asignificant increase in anti-F1 antibody titers when flagellin waspresent. These findings are analogous to those reported in Pasare andMedzhitov (2004) Immunity 21:733-741 using LPS as an adjuvant. Theauthors suggested that once CD4⁺ memory is established using LPS as anadjuvant, TLR stimulation is no longer required for activation of theselymphocytes. Although the memory response in our system remains to befully characterized, the lack of an F1-specific IgG response in athymicnude mice (BALB/cAnNCr-nu/nu) immunized with flagellin and F1 (FIG. 4,panel b) demonstrates a requirement for T cells in the humoral responseto this vaccine.

TNF-α, IL-6 and interferons are not required for the adjuvant effects offlagellin. Previously, it was determined that flagellin induces highlevels of TNF-α and IL-6 in the lung (Example 1; Honko and Mizel (2004)Infect. Immun. 72, 6676-6679). Therefore, the role of these cytokines inthe adjuvant activity of flagellin was evaluated. TNF-α is a pleiotropiccytokine that promotes dendritic cell maturation (Banchereau et al.(2000) Annu. Rev. Immunol. 18, 767-811). As shown in FIG. 5, panel a,the anti-F1 antibody response remains extremely high in these mice,indicating that TNF-α is not required for the adjuvant effect offlagellin. However, flagellin-stimulated TNF-α production appears toenhance the antibody response against F1 antigen, as the titers inTNFR^(−/−) mice were reduced approximately two-fold relative towild-type B6; 129 mice. The role of IL-6, a cytokine that promotes Bcell proliferation and differentiation (Kaminura et al. (2003) Rev.Physiol Biochem. Pharmacol. 149:1-38), in the adjuvant activity offlagellin was evaluated using IL-6^(−/−) mice. There was no defect inanti-F1 IgG production in these mice following immunization with F1 andFliC (FIG. 5, panel b), indicating that this cytokine is also notessential for the adjuvant effect of flagellin.

In vitro, flagellin stimulates nitric oxide and interferon-β (IFN-β)production via signaling through functional TLR5/4 heteromeric complexes(Mizel et al. (2003) J. Immunol 170:6217-6223). C3H/HeJ mice, whichpossess a nonfunctional mutant TLR4 (Poltorak et al. (1998) Science282:2085-2088), provide a model to separate the effects of TLR5/4heteromeric and TLR5/5 homomeric signaling in vivo. In the lung,flagellin-stimulated production of TNF-α, IL-6, G-CSF (granulocytecolony stimulating factor), keratinocyte-derived chemokine (KC),macrophage inflammatory protein 2 (MIP-2) and MIP-1α was not disruptedin C3H/HeJ mice (Example 1; Honko and Mizel (2004) Infect. Immun.72:6676-6679); however, interferon production was not evaluated. Type IIFNs are proposed to link innate and adaptive immune responses bystimulating the upregulation of MHC and costimulatory molecules onantigen-presenting cells (Le Bon and Tough (2002) Curr. Opin. Immunol.14:432-436). To determine the role of TLR5/4 signaling on the adjuvanteffect of flagellin, anti-F1 antibody titers in C3H/HeJ mice werecompared to their wild-type counterpart, C3H/HeN mice (FIG. 5, panel c),following immunization with F1 antigen and flagellin. As there was nodefect in antibody production, signaling via TLR5/4 complexes is notrequired for the adjuvant effects of flagellin. The role of interferonsin the adjuvant activity of flagellin was evaluated directly bydetermining antibody responses in mice lacking either type I interferonsignaling (IFNα/βR^(−/−)) or the ability to produce interferon-γ(IFNγ^(−/−)) (FIG. 5, panels d and e). As both strains of mice respondedin a manner similar to wild-type mice immunized with F1 antigen andflagellin, interferons are also not required for the adjuvant effect offlagellin.

Flagellin promotes a protective response for intranasal challenge withYersinia pestis CO92. The fundamental test of a vaccine is the abilityto provide protection against challenge with a pathogen. As a model forrespiratory infection, immunized and control mice were challengedintranasally with virulent Y. pestis CO92. To ensure that vaccinationinduced adequate protection, a challenge dose of 150 times the lethaldose 50% (LD₅₀) for i.n. infection with Y. pestis was selected on thebasis of recommendations from a recent Plague Vaccine Workshop sponsoredby the NIAID and the FDA (Oct. 13-14, 2004). BALB/c mice immunized andboosted i.n. with flagellin and F1 had a 93% survival rate, versus only7% in the control group (FIG. 6, panel a), following challenge with adose equivalent to 100×LD₅₀. B cell-deficient IgH^(−/−) mice were usedto evaluate the B cell/antibody dependence of the protective response(FIG. 6, panel b). All control and immunized mice succumbed to Y. pestisinfection at a dose of approximately 150×LD₅₀, indicating thatprotection at this challenge dose is B cell-mediated and thus presumablyantibody-mediated. Previously, Elvin et al. examined the role of type 1effector functions in protection from Y. pestis infection usingStat4^(−/−) animals that have defective IL-12 and IFN-γ-mediatedcellular immune responses (Elvin and Williamson (2004) Microb. Patho.37:177-184). Whereas immunized Stat4′-mice produced similar levels ofanti-F1 and anti-V IgG as their wild-type counterpart, these animalswere less protected from high dose challenge. To address the role ofIFN-γ-mediated protection following intranasal challenge in our system,groups of IFN-γ^(−/−) and wild-type C57BL/6 mice were immunized andboosted with F1 antigen and flagellin prior to challenge with 150×LD₅₀Y. pestis. Wild-type C57BL/6 mice were completely protected by i.n.immunization with flagellin and F1, versus only 10% of controls (FIG. 6,panel c). Immunized IFN-γ^(−/−) mice had 80% survival followingchallenge (FIG. 6, panel d), indicating that IFN-γ-mediated responsesare not required for protection. As these animals had high titers ofanti-F1 IgG, these results confirm the importance of F1-specificantibodies in protection from Y. pestis infection and support theutility of circulating IgG titers as a correlate of protective efficacy.The observation that two IFNγ^(−/−) mice succumbed to infection suggeststhat IFN-γ may augment antibody-mediated protection from Y. pestis,possibly through the promotion of the respiratory burst in phagocytes.

Flagellin is an effective adjuvant in nonhuman primates. In view of theability of flagellin to promote protective adaptive immune responses inmurine models, we next evaluated the effectiveness of flagellin as anadjuvant in nonhuman primates. A recombinant fusion protein consistingof the F1 and V antigens of Y. pestis was used for immunization offemale cynomolgus macaques. Groups of 6 monkeys were immunized with 150μg F1/V fusion and 50 μg flagellin i.n. or intramuscularly (i.m.).Additional control animals (n=3) received PBS by both routes. Prior toimmunization, the monkeys exhibited anti-flagellin antibody titers ofapproximately 9.8×10⁴. Monkeys immunized with flagellin exhibited nochange in body temperature or plasma TNF-α levels during the first 24 hfollowing immunization, and no observable inflammation occurred at thesite of injection. Animals were boosted in an identical manner at fourweeks and plasma anti-F1/V IgG titers were determined two weeks later(FIG. 7). Immunized monkeys exhibited a striking increase inF1/V-specific antibody titers. No antigen-specific IgE was detected.These results clearly establish that flagellin is an effective adjuvantfor the development of an antibody response in nonhuman primates, evenin the presence of circulating anti-flagellin antibodies.

Example 4 Yersinia pestis Antigens and Vaccines

A fusion protein to induce an immune response against Yersinia pestis isproduced in a like manner as described in Example 2, with a Y. pestis Vantigen, a Y. pestis F1 antigen, or a fusion peptide thereof. Suchfusion proteins can be used to induce an immune response, optionally aprotective immune response, as described herein. Optionally, theresponse is a mucosal immune response. Specific non-limiting examples ofsuitable fusion proteins are:

Example A: FliC/F1/V amino acid sequence (SEQ ID NO: 1) 1 MAQVINTNSLSLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI 51 ANRFTSNIKG LTQASRNANDGISIAQTTEG ALNEINNNLQ RVRELSVQAT 101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQTQFNGVKVLSQ DNQMKIQVGA 151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSFKNVTGRSMAD 201 LTASTTATAT LVEPARITLT YKEGAPITIM DNGNIDTELL VGTLTLGGYK251 TGTTSTSVNF TDAAGDPMYL TFTSQDGNNH QFTTKVIGKD SRDFDISPKV 301NGENLVGDDV VLATGSQDFF VRSIGSKGGK LAAGKYTDAV TVTVSNQGSI 351 EGRNRAYEQNPQHFIEDLEK VRVEQLTGHG SSVLEELVQL VKDKNIDISI 401 KYDPRKDSEV FANRVITDDIELLKKILAYF LPEDAILKGG HYDNQLQNGI 451 KRVKEFLESS PNTQWELRAF MAVMHFSLTADRIDDDILKV IVDSMNHHGD 501 ARSKLREELA ELTAELKIYS VIQAEINKHL SSSGTINIHDKSINLMDKNL 551 YGYTDEEIFK ASAEYKILEK MPQTTIQVDG SEKKIVSIKD FLGSENKRTG601 ALGNLKNSYS YNKDNNELSH FATTCSDKSR PLNDLVSQKT TQLSDITSRF 651NSAIEALNRF IQKYDSVMQR LLDDTSGK RS   ATGDK ITLAG KTMFIDKTAS 701GVSTLINEDA AAAKKSTANP LASIDSALSK VDAVRSSLGA IQNRFDSAIT 751 NLGNTVTNLNSARSRIEDAD YATEVSNMSK AQILQQAGTS VLAQANQVPQ 801 NVLSLLRLEH HHHHH*Example B: FliC/F1 amino acid sequence (SEQ ID NO: 2) 1 MAQVINTNSLSLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI 51 ANRFTSNIKG LTQASRNANDGISIAQTTEG ALNEINNNLQ RVRELSVQAT 101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQTQFNGVKVLSQ DNQMKIQVGA 151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSFKNVTGRSMAD 201 LTASTTATAT LVEPARITLT YKEGAPITIM DNGNIDTELL VGTLTLGGYK251 TGTTSTSVNF TDAAGDPMYL TFTSQDGNNH QFTTKVIGKD SRDFDISPKV 301NGENLVGDDV VLATGSQDFF VRSIGSKGGK LAAGKYTDAV TVTVSNQ RSA 351 TGDK ITLAGKTMFIDKTASG VSTLINEDAA AAKKSTANPL ASIDSALSKV 401 DAVRSSLGAI QNRFDSAITNLGNTVTNLNS ARSRIEDADY ATEVSNMSKA 451 QILQQAGTSV LAQANQVPQN VLSLLRLEHHHHHH* EXAMPLE C: FliC/V amino acid sequence (SEQ ID NO: 3) 1 MAQVINTNSLSLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI 51 ANRFTSNIKG LTQASRNANDGISIAQTTEG ALNEINNNLQ RVRELSVQAT 101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQTQFNGVKVLSQ DNQMKIQVGA 151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSFKNVTGRSMIR 201 AYEQNPQHFI EDLEKVRVEQ LTGHGSSVLE ELVQLVKDKN IDISIKYDPR251 KDSEVFANRV ITDDIELLKK ILAYFLPEDA ILKGGHYDNQ LQNGIKRVKE 301FLESSPNTQW ELRAFMAVMH FSLTADRIDD DILKVIVDSM NHHGDARSKL 351 REELAELTAELKIYSVIQAE INKHLSSSGT INIHDKSINL MDKNLYGYTD 401 EEIFKASAEY KILEKMPQTTIQVDGSEKKI VSIKDFLGSE NKRTGALGNL 451 KNSYSYNKDN NELSHFATTC SDKSRPLNDLVSQKTTQLSD ITSRFNSAIE 501 ALNRFIQKYD SVMQRLLDDT SGK RSATGDK  ITLAGKTMFIDKTASGVSTL 551 INEDAAAAKK STANPLASID SALSKVDAVR SSLGAIQNRF DSAITNLGNT601 VTNLNSARSR IEDADYATEV SNMSKAQILQ QAGTSVLAQA NQVPQNVLSL 651LRLEHHHHHH * Note: FliC obtained from S. enteritidis. In each of thesefusion proteins, the N terminal constant region of FliC ends at aminoacid residue 198, and the C terminal constant region (the first sevenamino acids of which is denoted in bold and underlined) begins at aminoacid residue 679 of Example A (SEQ ID NO: 1), amino acid residue 348 ofExample B (SEQ ID NO: 2), and amino acid residue 524 of Example C (SEQID NO: 3).

Example 5 Biological Activity of a Fusion Peptide Containing Flagellinand the F1 and V Antigens of Yersinia pestis

To prepare an expression plasmid encoding flagellin and Yersinia pestisF1 and V antigens as a single protein, the majority of the nucleotidesequence encoding the hypervariable region of the S. enteritidisflagellin was removed and replaced with the F1 and V sequences in tandemseparated by an eighteen nucleotide bridge encoding 6 amino acids (seeExample A, SEQ ID NO:1, above). The recombinant protein was produced inBL21 cells and purified by affinity chromatography on a metal affinityresin. Endotoxin and contaminating nucleic acid were removed using anAcrodisc chromatography filter. To determine if the resultant proteinretained flagellin bioactivity, TLR5-negative and TLR5-positive RAW264.7cells were incubated with the trifusion protein and the extent of tumornecrosis factor-a production was determined. The TLR5-negative RAW cellswere used to control for any contaminating factors that might have aneffect in this assay. As shown in FIG. 8, a fusion protein containingflagellin and the Yersinia pestis F1 and V proteins retains flagellinbiological activity in TLR5-positive cells. The protein did not signalin TLR5-negative RAW264.7 cells.

Toll-like receptor 5 (TLR5)-negative RAW264.7 cells or TLR5-positiveRAW264.7 cells (a cell line created by stably transfecting RAW264.7cells with a construct encoding a TLR5-enhanced yellow fluorescentprotein) were incubated with increasing concentrations of a fusionprotein encoding flagellin and the F1 and V proteins of Y. pestis for 4hours and then the culture medium was assayed by ELISA for the contentof TNF-α

To determine if flagellin+F1+V or a single protein containing all threeprotects against a lethal challenge with Y. pestis CO92, C3H/HeJ micewere immunized and boosted with phosphate buffered saline only (PBS) ora vaccine containing three proteins-i mg flagellin+5 mg each F1 and V ora vaccine containing a single protein containing flagellin, F1, and V(flagellin/F1V; 10 mg). The mice were boosted after 4 weeks with thesame regimens and then challenged with approximately 150 LD₅₀ of Y.pestis CO92. The mice were bled prior to the challenge and anti-F1 IgGtiters were determined by ELISA. As shown in Table 2, flagellin+F1 and Vantigens of Yersinia pestis or a fusion protein containing flagellin andthe F1 and V antigens of Yersinia pestis provides complete protectionagainst a lethal respiratory challenge with Yersinia pestis.

TABLE 2 Results of protection studies of mice immunized with flagellin +F1 and V antigens of Y. pestis or a fusion protein containing flagellinand the F1 and V antigens of Y. pestis against a lethal respiratorychallenge with Y. pestis. Survival Immunization Anti-F1 IgG titer Anti-VIgG titer Number Percent PBS 6.6 × 10² 1.2 × 10³  0/10 0 Flagellin + 4.5× 10⁶ 4.2 × 10⁶ 10/10 100 F1 + V Flagellin/F1/V   5 × 10⁶ 6.3 × 10⁶ 4/4100

The foregoing is illustrative of the present invention, and is not to betaken as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A fusion protein comprising: (a) a flagellin adjuvant comprising: (i)a flagellin N-terminal constant region; and (ii) a flagellin C-terminalconstant region; and (b) a tumor antigen between the N-terminal constantregion and the C-terminal constant region.
 2. The fusion protein ofclaim 1, wherein the flagellin adjuvant comprises a deleted flagellinhypervariable region or the flagellin hypervariable region is absentfrom the flagellin adjuvant.
 3. The fusion protein of claim 1, whereinthe tumor antigen is inserted (i) within the hypervariable region, (ii)between the flagellin N-terminal constant region and the hypervariableregion, or (iii) between the flagellin C-terminal constant region andthe hypervariable region.
 4. The fusion protein of claim 1, wherein thetumor antigen is a breast tumor antigen.
 5. The fusion protein of claim1, wherein the tumor antigen is a cancer/testis antigen.
 6. A nucleicacid encoding the fusion protein of claim
 1. 7. A vector comprising thenucleic acid of claim
 6. 8. A host cell comprising the nucleic acid ofclaim
 6. 9. A method of making the fusion protein, the method comprisingculturing the host cell of claim 8 in a culture medium under conditionssufficient for the fusion protein to be produced.
 10. The method ofclaim 9, wherein the fusion protein is collected from the host cell orfrom the culture medium.
 11. An immunogenic composition comprising thefusion protein of claim 1 in a pharmaceutically acceptable carrier. 12.A method of producing an immune response against a tumor antigen in amammalian subject, the method comprising administering the immunogeniccomposition of claim 11 to the mammalian subject in an amount effectiveto produce an immune response in the mammalian subject against the tumorantigen.
 13. A method of treating a tumor in a mammalian subject, themethod comprising administering the immunogenic composition of claim 11to the mammalian subject in a treatment effective amount.
 14. The methodof claim 13, wherein the tumor is a breast tumor.
 15. An immunogeniccomposition comprising in a pharmaceutically acceptable carrier: (a) aflagellin adjuvant; and (b) a tumor antigen.
 16. The immunogeniccomposition of claim 15, wherein the tumor antigen is a breast tumorantigen.
 17. The immunogenic composition of claim 15, wherein the tumorantigen is a cancer/testis antigen.
 18. The immunogenic composition ofclaim 17, wherein the immunogenic composition comprises two or morecancer/testis antigens.
 19. The immunogenic composition of claim 15,wherein the tumor antigen is coupled to the flagellin adjuvant.
 20. Amethod of producing an immune response against a tumor antigen in amammalian subject, the method comprising administering the immunogeniccomposition of claim 15 to the mammalian subject in an amount effectiveto produce an immune response against the tumor antigen in the mammaliansubject.
 21. A method of treating a tumor in a mammalian subject, themethod comprising administering the immunogenic composition of claim 15to the mammalian subject in a treatment effective amount.
 22. The methodof claim 21, wherein the tumor is a breast tumor.